Epilepsy, Behavioral Abnormalities, and Physiological Comorbidities in Syntaxin-Binding Protein 1 (STXBP1) Mutant Zebrafish.

Mutations in the synaptic machinery gene syntaxin-binding protein 1, STXBP1 (also known as MUNC18-1), are linked to childhood epilepsies and other neurodevelopmental disorders. Zebrafish STXBP1 homologs (stxbp1a and stxbp1b) have highly conserved sequence and are prominently expressed in the larval zebrafish brain. To understand the functions of stxbp1a and stxbp1b, we generated loss-of-function mutations using CRISPR/Cas9 gene editing and studied brain electrical activity, behavior, development, heart physiology, metabolism, and survival in larval zebrafish. Homozygous stxbp1a mutants exhibited a profound lack of movement, low electrical brain activity, low heart rate, decreased glucose and mitochondrial metabolism, and early fatality compared to controls. On the other hand, homozygous stxbp1b mutants had spontaneous electrographic seizures, and reduced locomotor activity response to a movement-inducing "dark-flash" visual stimulus, despite showing normal metabolism, heart rate, survival, and baseline locomotor activity. Our findings in these newly generated mutant lines of zebrafish suggest that zebrafish recapitulate clinical phenotypes associated with human syntaxin-binding protein 1 mutations

Morphology of <i>stxbp1a</i>^s3000 mutant zebrafish.

<p>(A) Heterozygous <i>stxbp1a</i>^{<i>s3000/+</i>} mutant larvae (5 dpf) are morphologically indistinguishable from wild-type siblings. Homozygous <i>stxbp1a</i>^{<i>s3000/s3000</i>} mutant larvae are immobile and fail to hatch out of the chorion. Scale bar = 500 μm. (B) Homozygous <i>stxbp1a</i>^{<i>s3000/s3000</i>} mutant larvae (n = 10) removed from their chorions are not significantly different in length from their siblings (n = 30; p = 0.0592, two-tailed t-test). (C-D) At 5 dpf, the dorsal surface of homozygous <i>stxbp1a</i>^{<i>s3000/s3000</i>} mutant larvae that were removed from their chorions at 2 dpf (D) show dispersed melanin and foreshortened craniofacial structure compared to siblings (C). Scale bar = 300 μm.</p

Zebrafish <i>stxbp1a</i> CRISPR/Cas9 mutant allele.

<p>(A) Sites of human and zebrafish mutations in highly conserved Stxbp1 sequence. Human STXBP1A protein sequence was aligned to zebrafish Stxbp1a and Stxbp1b. Black background indicates amino acid residues that are similar in all three proteins (BLOSUM 62). Grey background indicates amino acid residues that are similar between two of the three proteins. The salmon-colored background indicates the position of the deletion mutation in zebrafish mutant alleles. (B) Alignment of the mutant <i>stxbp1a</i>^s3000 allele sequence (top) with wild-type zebrafish <i>stxbp1a</i> sequence (bottom). The CRISPR/Cas9 target site is shown as a wide purple arrow below the plot. The site of the 4bp deletion <i>stxbp1a</i>^s3000 allele is highlighted in salmon, and corresponds to amino acids 211–212 highlighted in (A). (C) Alignment of the mutant <i>stxbp1b</i>^<i>s3001</i> allele sequence with wild-type zebrafish <i>stxbp1b</i> sequence.</p

Ontogeny of <i>stxbp1a</i> and <i>stxbp1b</i> expression in zebrafish larvae.

<p>Wild-type zebrafish were probed for expression of <i>stxbp1a</i> mRNA (A-E) or <i>stxbp1b</i> mRNA (F-J). Abbreviations: CeP: cerebellar plate, Ha: habenula, IR: inner retina, MO: medulla oblongata, OB: olfactory bulb, P: pallium, Ret: retina, SC: spinal cord, Tel: telencephalon, TeO: optic tectum, Scale bar = 100μm.</p

Dark-flash response deficit in <i>stxbp1b</i> mutant zebrafish larvae.

<p>(A) Normal mobility in homozygous <i>stxbp1b</i>^<i>-/-</i> mutant larval zebrafish. Cumulative plots of the position and velocity of 10 representative wild-type larvae, 10 representative <i>stxbp1b</i>^{<i>s3001/+</i>} heterozygous mutants, and 10 <i>stxbp1b</i>^{<i>s30010/s3001</i>} homozygous mutants during 10 minutes of behavioral recording. Larval zebrafish (5 dpf) were placed in individual wells of a flat-bottom 96-well plate and acclimated to the Daniovision recording chamber before tracking began. Yellow indicates low velocity movement; red indicates high velocity movement. Scale bar = 1 cm. (B) Larval zebrafish (5 dpf) were placed in individual wells of a flat-bottom 96-well plate and acclimated to the Daniovision recording chamber. 24 hours of movement data were collected beginning at 4:00 PM. Data shown are sums of 10-minute bins (mean ± SD; n = 13 WT, 23 Het, 12 Mut). (C) Homozygous <i>stxbp1b</i>^{<i>s3001/s3001</i>} and heterozygous <i>stxbp1b</i>^{<i>s3001/+</i>} mutants’ baseline movement did not differ statistically from wild-type baseline movement. There were no significant differences in movement (seconds spent moving) between heterozygous <i>stxbp1b</i>^{<i>s3001/+</i>} and wild-type siblings (two-tailed t-tests, n = 13 WT, 23 Het, 12 Mut), or for velocity or distance traveled (not shown; mean ± SD). (D) In response to transition from 100% light intensity to darkness (0% light intensity), homozygous <i>stxbp1b</i>^{<i>s3001/s3001</i>} larvae (6 dpf) responded with less movement than either their wild-type siblings (two-tailed t-test, p = 0.021) or their heterozygous siblings (two-tailed t-test, p = 0.00024) mean ± SE (n = 13 WT, 23 Het, 12 Mut).</p

Metabolic and survival deficits caused by <i>stxbp1a</i>^s3000 mutation.

<p>Homozygous <i>stxbp1a</i>^{<i>s3000/s3000</i>} mutant larvae have lower metabolism than controls (siblings) and die prematurely. (A) 5 dpf <i>stxbp1a</i>^{<i>s3000/s3000</i>} homozygous mutants (n = 10) had significantly lower extracellular acidification (ECAR) than siblings (n = 10; mean ± SD). * = p < 0.0001 (two-tailed t-test). (B) 5 dpf <i>stxbp1a</i>^{<i>s3000/s3000</i>} homozygous mutants (n = 10) had significantly lower oxygen consumption (OCR) than siblings (n = 10; mean ± SD). * = p < 0.0001 (two-tailed t-test). (C) Heart rates of <i>stxbp1a</i>^{<i>s3000/s3000</i>} mutant larvae (n = 15) at 3 dpf were significantly lower than heart rates of their siblings (n = 33). All measured values are plotted; mean values are indicated by horizontal lines. (D) Homozygous <i>stxbp1a</i>^{<i>s3000/s3000</i>} mutants die as larvae. Homozygous mutants (n = 50) and siblings (n = 50) were maintained in petri dishes without food and counted each day from 2 dpf until 10dpf. The <i>stxbp1a</i>^{<i>s3000/s3000</i>} mutants began dying at 6 dpf, and 98% (49) died by 10dpf. Only 2% (1) of siblings died at 10dpf.</p

Normal metabolic function in <i>stxbp1b</i>^<i>s3001</i> mutants.

<p>Homozygous <i>stxbp1b</i>^{<i>s30010/s3001</i>} mutant larvae have unaffected metabolism compared to controls (siblings). (A) 5 dpf <i>stxbp1b</i>^{<i>s30010/s3001</i>} homozygous mutants (n = 11) did not differ from siblings (n = 52) in extracellular acidification (ECAR) (two-tailed t-test; mean ± SD). (B) 5 dpf <i>stxbp1b</i>^{<i>s30010/s3001</i>} homozygous mutants (n = 11) did not differ from siblings (n = 52) in oxygen consumption (OCR) (two-tailed t-test; mean ± SD). (C) Heart rates of <i>stxbp1b</i>^{<i>s30010/s3001</i>} mutant larvae (n = 16) at 3 dpf were not significantly different from heart rates of their siblings (n = 34). All measured values are plotted; mean values are indicated by horizontal lines. (D) All <i>stxbp1b</i>^{<i>s30010/s3001</i>} mutant larvae tested (n = 8) survived until 10 dpf; 12.5% of their control siblings (n = 48) died by 10dpf.</p

Electrophysiological phenotypes of <i>stxbp1a</i> and <i>stxbp1b</i> homozygous mutant larvae.

<p>(A) Forebrain field potential recordings from homozygous <i>stxbp1a</i>^{<i>s3000/s3000</i>} and <i>stxbp1b</i>^{<i>s3001/s3001</i>} mutant larvae compared to a control recording from an electrode in 1.2% low-melting point agarose or age-matched wild-type zebrafish; approximately 1 min of gap-free recordings are shown. (B) Magnified view of the yellow highlighted region in the above trace from a <i>stxbp1b</i>^{<i>s3001/s3001</i>} mutant larva. (C) Mean duration of all spontaneous events (greater than 50 msec in duration) was significantly greater in <i>stxbp1b</i>^{<i>s3001/s3001</i>} mutant larvae compared to <i>stxbp1a</i>^{<i>s3000/s3000</i>} larvae, WT or agar; *p < 0.001 One-way ANOVA on ranks with a Dunn’s multiple comparison test. (D) Frequency of spontaneous events was also significantly greater in <i>stxbp1b</i>^{<i>s3001/s3001</i>} mutant larvae compared to WT or agarose; *p < 0.001 One-way ANOVA on ranks with a Dunn’s multiple comparison test.</p

Behavior in double mutant <i>stxbp1a</i>^{<i>s3000/+</i>} <i>stxbp1b</i>^{<i>s3001/+</i>} larvae.

<p>Graph of distance traveled per ten-minute interval by 5dpf wildtype (WT, n = 16) and <i>stxbp1a</i>^{<i>s3000/+</i>} <i>stxbp1b</i>^{<i>s3001/+</i>} double mutant zebrafish (AB, n = 24) larvae over 24 hours. Mean distance per 10-min interval was 479.3 for WT larvae and 445.4 for AB larvae, significantly different by ANOVA and Tukey post hoc tests (p<0.001) as described in the main text.</p

Morphology of <i>stxbp1b</i>^<i>s3001</i> mutant zebrafish.

<p>(A) Both heterozygous <i>stxbp1b</i>^{<i>s3001/+</i>} and homozygous <i>stxbp1b</i>^{<i>s3001/s3001</i>} mutant larvae (5 dpf) are morphologically similar to their wild-type siblings. Scale bar = 500 μm. (B) Homozygous <i>stxbp1b</i>^{<i>s3001/s3001</i>} mutant larvae (n = 8) are not significantly different in length from their siblings (n = 13) (p = 0.2297, two-tailed t-test). (C-D) The dorsal surface of homozygous <i>stxbp1b</i>^{<i>s3001/s3001</i>} mutant larvae (D) show dispersed melanin compared to siblings (C). Scale bar = 300 μm.</p